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Results for “regulatory element transcripts”

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PubMed1993

Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene.

The human Na,K-ATPase beta 1 subunit gene promoter activity is stimulated by thyroid hormone (T3) in the human intestinal Caco-2 cells. To identify potential cis-acting transcriptional regulatory elements involved in this process, chimeric plasmids containing varying lengths of the 5' flanking region of the human beta 1 Na,K-ATPase gene linked to the firefly luciferase reporter gene were introduced into Caco-2 cells by transient transfection. Analysis of T3-regulated luciferase activity of cells carrying these plasmids, and subsequent use of site-directed mutagenesis revealed that a region from -459 to -438 (relative to the transcriptional start site) is required for the induction of the beta 1 Na,K-ATPase gene by T3. An oligonucleotide containing this sequence from -465 to -433 confers T3 responsiveness to a heterologous promoter. Gel mobility shift assays showed specific binding of nuclear proteins of Caco-2 cells to this region and immunoreactive T3 receptor was identified in one of these complexes. These data demonstrate that there is a cis-acting thyroid hormone responsive element in the 5' flanking region of the human beta 1 Na,K-ATPase gene and induction of transcription of this gene by T3 involves specific binding of the thyroid hormone receptor to the TRE located at position -459 to -438.

Adenocarcinoma↗

PubMed2006

An ARC light on lipid metabolism.

The SREBP pathway plays a central role in the regulation of lipid metabolism. In a recent letter, Yang et al. present a comprehensive series of experiments, spanning a wide range of disciplines, that identify ARC105 as a component of the ARC complex that interacts directly with SREBP and is necessary for SREBP function (Yang et al., 2006).

Animals↗

PubMed1997

Physical interactions between Ets and NF-kappaB/NFAT proteins play an important role in their cooperative activation of the human immunodeficiency virus enhancer in T cells.

The transcriptional regulatory elements of many inducible T-cell genes contain adjacent or overlapping binding sites for the Ets and NF-kappaB/NFAT families of transcription factors. Similar arrays of functionally important NF-kappaB/NFAT and Ets binding sites are present in the transcriptional enhancers of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2), suggesting that this pattern of nuclear protein binding sites reflects an evolutionarily conserved mechanism for regulating inducible T-cell gene expression that has been co-opted during HIV evolution. Despite these findings, the molecular mechanisms by which Ets and NF-kappaB/NFAT proteins cooperatively regulate inducible T-cell gene expression remained unknown. In the studies described in this report, we demonstrated a physical interaction between multiple Ets and NF-kappaB/NFAT proteins both in vitro and in activated normal human T cells. This interaction is mediated by the Ets domain of Ets proteins and the C-terminal region of the Rel homology domains of NF-kappaB/NFAT proteins. In addition, the Ets-NF-kappaB/NFAT interaction requires the presence of DNA binding sites for both proteins, as it is abolished by the DNA intercalating agents propidium iodide and ethidium bromide and enhanced by the presence of synthetic oligonucleotides containing binding sites for Ets and NF-kappaB proteins. A dominant-negative mutant of NF-kappaB p50 that binds DNA but fails to interact with Ets proteins inhibits the synergistic activation of the HIV-1 and HIV-2 enhancers by NF-kappaB (p50 + p65) and Ets-1, suggesting that physical interaction between Ets and NF-kappaB proteins is required for the transcriptional activity of the HIV-1 and HIV-2 enhancers. Taken together, these findings suggest that evolutionarily conserved physical interactions between Ets and NF-kappaB/NFAT proteins are important in regulating the inducible expression of T-cell genes and viruses. These interactions represent a potential target for the development of novel immunosuppressive and antiviral therapies.

Base Sequence↗

PubMed2001

Quantitative real-time reverse transcription-PCR assay for cyclin D1 expression: utility in the diagnosis of mantle cell lymphoma.

BACKGROUND: The t(11;14)(q13;q32) translocation present in the majority of mantle cell lymphomas (MCLs) places the cyclin D1 gene under the control of immunoglobulin transcriptional regulatory elements, causing overexpression of cyclin D1. Quantification of cyclin D1 expression can distinguish MCL from other lymphomas. METHODS: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, beta(2)-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, M02058. RESULTS: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes. CONCLUSIONS: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL.

Blotting, Northern↗

PubMedDate not supplied

Regulation of LDL receptor expression by the effect of curcumin on sterol regulatory element pathway.

To investigate the molecular mechanisms of the effect of curcumin on up-regulation of LDL receptor expression, a sterol regulatory report system was established in Xenopus laevis oocytes by microinjection of a plasmid pLXRN-4SRE-fPA, in which green fluorescence protein (GFP) gene was constructed downstream from the sterol regulatory element-1 (SRE-1), in the nucleus of the oocytes. The oocytes were treated with different kinds of natural medicines: curcumin, tea polyphenols, tanshinone, ligustrazine, ginkgolide, astragalosides. The expression of GFP was induced only by curcumin. In addition, it was also proved that the curcumin can increase the expression of functional LDL receptors in human liver hepatoma cell line HepG2. In conclusion, curcumin may up-regulate the expression of LDL receptor by its effect on SRE pathway.

Animals↗

PubMed1998

Characterization of a polypurine/polypyrimidine sequence upstream of the mouse metallothionein-I gene.

A 128 base pair long homopurine/homopyrimidine (R/Y) element is located approximately 1.2 kb upstream of the transcription start point of the mouse metallothionein-I ( MT-I ) gene. We present a detailed in vitro structural characterization of the MT-I R/Y sequence as determined by enzymatic and chemical probes. An approximately 190 bp fragment containing the MT-I R/Y sequence was subcloned into a recombinant vector. Low resolution analysis with S1 nuclease indicates that DNA in this region was unpaired in supercoiled plasmids treated at low pH. High resolution mapping with chemical probes selective for non-B DNA structures provides evidence that the MT-I R/Y sequence adopts one or more H-DNA structures. We also investigated this sequence to determine if it can influence transcriptional regulation. Promoter/reporter constructs were prepared in which the MT-I R/Y sequence was positioned in either orientation upstream of either the MT-I or HSV-TK promoters. Promoter/reporter activities were evaluated by transient transfection assays using mouse NIH3T3 cells. The MT-I R/Y sequence displayed no detectable activity as a cis -acting transcriptional regulatory element. These results demonstrate that although the MT-I R/Y sequence is able to adopt a non-B DNA structure under certain in vitro conditions, there is no evidence that this sequence plays a significant role in transcriptional regulation.

3T3 Cells↗

PubMed1988

Strong transcriptional activation of translocated c-myc genes occurs without a strong nearby enhancer or promoter.

We have studied the transcriptional activation of translocated c-myc genes in murine plasmacytomas in which the translocation juncture occurs within the first intron of c-myc and juxtaposes c-myc with the immunoglobulin C alpha gene segment. It has been widely suggested that a novel transcriptional enhancer element located near the C alpha gene segment might activate the translocated c-myc gene. We have carried out an extensive search for such an element and find no significant transcriptional enhancer activity in a 22 kb region encompassing the translocation junction, C alpha gene segment and regions 3' of C alpha. We also find that the cryptic promoter region of the translocated c-myc gene is a very weak promoter of transcription. Despite this evidence against the presence of strong transcriptional regulatory elements, the translocated c-myc gene locus is transcribed at high rates that are 25-greater than 100% of that measured for the highly active immunoglobulin genes in murine plasmacytomas. These data suggest the presence of a novel type of strong activator of transcription in the murine heavy chain locus.

Animals↗

PubMed1995

A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but was absent from mammary gland cells. These results suggest that a factor contributing to high-level expression of MMTV in the mammary gland is the lack of negative regulation by NBP.

Animals↗

PubMed1994

Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene.

Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.

Amino Acid Sequence↗

PubMed1993

5' distal and proximal cis-acting regulator elements are required for developmental control of a rice seed storage protein glutelin gene.

Using a homologous transgenic rice system it is demonstrated that 5' distal and proximal cis-acting transcriptional regulatory elements are required for developmental control of a rice seed storage protein glutelin gene. Analyses of gene expression of nine progressively truncated 5' promoter sequences in developing endosperm indicated the existence of at least one major positive element located from the -5.1 to -1.8 kb region. The functional importance of proximal elements in the context of 1.8 kb promoter was demonstrated by single substitution mutations in the TATA box (-28/-23), AACA motif (-73/-61), and protein-binding boxes I (-103/-86), II (-124/-110), III (-175/-158) and IV (-200/-217). A simultaneous mutation of five protein-binding sites (-410/-86) essentially eliminated the activity of the 1.8 kb promoter. Although temporal control of the Gt1 gene during endosperm development was retained in plants of constructs from -5.1 kb to -155 bp, spatial control of the glutelin gene was altered when the 5.1 kb promoter was deleted to -507 bp or -154 bp as the reporter gene activities of these constructs were detected in phloem of leaves, and in stems, sheaths and roots of plants.

Base Sequence↗

PubMed2000

Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions.

The three distal transcriptional regulatory elements of the rat pyruvate kinase M gene, referred to as boxes A, B, and C, are located around -270 base pairs upstream from the transcriptional initiation site. Electrophoretic mobility shift assays with specific competitors and antibodies show that both box A and box B bind to Sp1 and Sp3 and that box C binds nuclear factor-Y (NF-Y). Luciferase reporter assays revealed that although box A and box B alone have no independent effect on luciferase activities, box C alone stimulates transcription. However, the inclusion of all three elements lead to maximal activity because of a synergistic effect, mainly between box B and box C, suggesting that functional synergism between Sp1/Sp3 and NF-Y is critical for the pyruvate kinase M (PKM) gene distal promoter activity. In fact, co-transfection of a dominant negative mutant of NF-YA (NF-YA29) resulted in a decrease in reporter activity in a box C-dependent manner. In addition, the overexpression of Sp1 or Sp3 and NF-Y in Drosophila SL2 cells synergistically stimulated PKM gene distal promoter activity. Using a mammalian two-hybrid system in HeLa cells, it was shown that both Sp1 and Sp3 interacted with NF-YA but not NF-YB and NF-YC. Moreover, glutathione S-transferase pull-down assays revealed that only in vitro translated (35)S-labeled NF-YA interacted with both Sp1 and Sp3 in vitro. A subunit interaction domain of NF-YA, which forms a heterotrimer with NF-YB and NF-YC, is not required for these interactions with Sp1 or Sp3. Thus, we conclude that Sp1, Sp3, and NF-Y stimulate the transcription of the PKM gene via their interactions.

Animals↗

PubMed2005

Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome.

Transcriptional promoters comprise one of many classes of eukaryotic transcriptional regulatory elements. Identification and characterization of these elements are vital to understanding the complex network of human gene regulation. Using full-length cDNA sequences to identify transcription start sites (TSS), we predicted more than 900 putative human transcriptional promoters in the ENCODE regions, representing a comprehensive sampling of promoters in 1% of the genome. We identified 387 fragments that function as promoters in at least one of 16 cell lines by measuring promoter activity in high-throughput transient transfection reporter assays. These positive functional results demonstrate widespread use of alternative promoters. We show a strong correlation between promoter activity and the corresponding endogenous RNA transcript levels, providing the first experimental quantitative estimate of promoter contribution to gene regulation. Finally, we identified functional regions within a randomly selected subset of 45 promoters using deletion analyses. These experiments showed that, on average, the sequence -300 to -50 bp of the TSS positively contributes to core promoter activity. Interestingly, putative negative elements were identified -1000 to -500 bp upstream of the TSS for 55% of genes tested. These data provide the largest and most comprehensive view of promoter function in the human genome.

Gene Expression Regulation↗

PubMed2005

Stability regulation of mRNA and the control of gene expression.

Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. Standard techniques measure changes in total cellular poly(A) mRNA levels. The assumption that changes in gene expression as measured by these techniques are directly and well correlated with changes in rates of new gene synthesis form the basis of attempts to connect coordinated changes in gene expression with shared transcription regulatory elements. Yet systematic attempts at this approach remain difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Recent technical advances have led to the successful scale-up and application of nuclear run-on procedures directly to microarrays. This development has allowed a gene-by-gene comparison between new gene synthesis in the nucleus and measured changes in total cellular polyA mRNA. Results from these studies have begun to challenge the strict interpretation of changes in gene expression measured by conventional microarrays as being closely correlated with changes in mRNA transcription rate, but rather they tend to support the significant expansion of the role played by changes in mRNA stability regulation to standard analyses of gene expression. Gene expression profiles obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in total cellular polyA mRNA in this system. Stability regulation was inferred by the absence of corresponding regulation of nuclear gene transcription activity for groups of genes strongly regulated at the whole cell level and which were also resistant to inhibition by Actinomycin D pre-treatment. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. It is proposed that the regulation of mRNA stability in response to external stimuli contributes significantly to observed changes in gene expression as measured by high throughput systems.

Cell Line, Tumor↗

PubMed2001

Heat shock regulatory elements are present in telomeric repeats of Chironomus thummi.

As in other Diptera, the telomeres of Chironomus thummi lack canonical short telomerase-specified repeats and instead contain complex sequences. They react to heat shock and other stress treatments by forming giant puffs at some chromosome termini, which are visible in polytene cells. All telomeres, except the telocentric end of chromosome four (4L), consist of large blocks of repeats, 176 bp in length. Three subfamilies of telomeric sequences have been found to show different distribution patterns between chromosome ends. TsA and TsC are characteristic of telomeres 3R and 4R, respectively, whereas TsB is present in the other non-telocentric telomeres. Heat shock transcription regulatory elements have been identified in the telomeric sequences, appearing differentially represented in the three subfamilies, but otherwise rather similar in size and sequence. Interestingly, TsA and TsB repeats share the well-conserved heat shock element (HSE) and GAGA motif, while the TATA box is only present in the former. Neither a HSE nor a TATA box appear in TsC repeats. Moreover, experimental data indicate that the HSE is functionally active in binding heat shock transcription factor (HSF). These results provide, for the first time, a molecular basis for the effect of heat shock on C.thummi telomeres and might also explain the different behaviour they show. A positive correlation between the presence of HSE and telomeric puffing and transcription under heat shock was demonstrated. This was also confirmed in the sibling species Chironomus piger. The significance of heat shock activation of telomeric repeats in relation to telomeric function is unknown at present, but it might be compared to the behaviour of other non-heat shock protein coding sequences, such as SINE-like and LINE-like retroelements, which have been reported to be activated by stress.

Animals↗

PubMed1989

Importance of introns for expression of mouse ribosomal protein gene rpL32.

The importance of intronic sequences for expression of the mouse ribosomal protein gene rpL32 was evaluated by transfection experiments with a series of mutant constructs in which one or more of the three rpL32 introns was totally or partially deleted. When transiently transfected into monkey kidney (COS) cells or stably transfected into mouse L cells, a mutant that lacked all three introns was completely inactive. Constructs that contained intron 1, either alone or in combination with another intron, were expressed as efficiently as was the normal intact rpL32 gene. Constructs that lacked intron 1 but contained another spliceable intron, even one from a foreign gene, were expressed at about 10 to 20% of the maximum level. These results indicated that intron 1 contains an element that increases the level of expression by 5- to 10-fold. A comparison of internal deletion mutants localized the element to within the first 27 base pairs of intron 1. Nuclear run-on experiments with stably transfected COS cells demonstrated that this element functions at the transcriptional level. The element was inactive when translocated to a position upstream of the transcriptional start site or to a position within intron 3, which indicated that it does not have the properties of a typical enhancer. From these and other results, we conclude that introns have both a general and a specific role in rpL32 expression. The general role, which can be satisfied by any spliceable intron, is to ensure an efficient yield of RNA transcripts. The specific role is uniquely attributable to intron 1, which contains a transcriptional regulatory element near its 5' end.

Animals↗

PubMed2001

Characterization of mouse Atp6i gene, the gene promoter, and the gene expression.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-type ATPase. We previously cloned a gene encoding a putative osteoclast-specific proton pump subunit, termed OC-116 kDa, approved mouse Atp6i (ATPase, H+ transporting, [vacuolar proton pump] member I). The function of Atp6i as osteoclast-specific proton pump subunit was confirmed in our mouse knockout study. However, the transcription regulation of Atp6i remains largely unknown. In this study, the gene encoding mouse Atp6i and the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of Atp6i have been characterized. Intrachromosomal mapping studies revealed that the gene contains 20 exons and 19 introns spanning approximately 11 kilobases (kb) of genomic DNA. Alignment of the mouse Atp6i gene exon sequence and predicted amino acid sequence to that of the human reveals a strong homology at both the nucleotide (82%) and the amino acid (80%) levels. Primer extension assay indicates that there is one transcription start site at 48 base pairs (bp) upstream of the initiator Met codon. Analysis of 4 kb of the putative promoter region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements. Northern blot analysis of RNAs from a number of mouse tissues reveals that Atp6i is expressed predominantly in osteoclasts, and this predominant expression was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) assay and immunohistochemical analysis. Whole-mount in situ hybridization shows that Atp6i expression is detected initially in the headfold region and posterior region in the somite stage of mouse embryonic development (E8.5) and becomes progressively restricted to anterior regions and the limb bud by E9.5. The expression level of Atp6i is largely reduced after E10.5. This is the first report of the characterizations of Atp6i gene, its promoter, and its gene expression patterns during mouse development. This study may provide valuable insights into the function of Atp6i, its osteoclast-selective expression, regulation, and the molecular mechanisms responsible for osteoclast activation.

Adenosine Triphosphatases↗

PubMed1999

Dynamic visualization of nervous system in live Drosophila.

We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5' flanking DNA of the Drosophila Na(+), K(+)-ATPase beta-subunit gene Nervana2 (Nrv2). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS-GFP (S65T) transgenes (Nrv2-GAL4+UAS-GFP) inserted on the same chromosomes. We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults. The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila. These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.

Animals↗

PubMed2004

Complementation of melanocyte development in SOX10 mutant neural crest using lineage-directed gene transfer.

An in vitro gene complementation approach has been developed to dissect gene function and regulation in neural crest (NC) development and disease. The approach uses the avian RCAS virus to express genes in NC cells derived from transgenic mice expressing the RCAS receptor TVA, under the control of defined promoter elements. Constructs for creating TVA transgenic mice were developed using site-specific recombination GATEWAY (GW), compatible vectors that can also be used to facilitate analysis of genomic fragments for transcriptional regulatory elements. By using these GW vectors to facilitate cloning, transgenic mouse lines were generated that express TVA in SOX10-expressing NC stem cells under the control of the Pax3 promoter. The Pax3-tv-a transgene was bred onto a Sox10-deficient background, and the feasibility of complementing genetic NC defects was demonstrated by infecting the Pax3-tv-a cells with an RCAS-Sox10 expression virus, thereby rescuing melanocyte development of Sox10-deficient NC cells. This system will be useful for assessing genetic hierarchies in NC development. Developmental Dynamics 229:54-62, 2004.

Animals↗